The present invention relates to the field of combinatorial libraries. More specifically, the invention relates to methods of synthesis utilizing arrays of solid-phase supports to produce a combinatorial library of chemical compounds and, additionally, the apparatuses used to carry out those methods.
Citation or identification of any reference in section 2 or any section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
A combinatorial library is a collection of multiple species of chemical compounds comprised of smaller subunits or monomers. Combinatorial libraries come in a variety of sizes, ranging from a few hundred to several thousand species of chemical compounds. There are also a variety of library types, including oligomeric and polymeric libraries comprised of compounds such as peptides, carbohydrates, oligonucleotides, and small organic molecules, etc. Such libraries have a variety of uses, such as identifying and characterizing ligands capable of binding an acceptor molecule or mediating a biological activity of interest.
The library compounds may comprise any type of molecule of any type of subunits or monomers, including polymers wherein the monomers are chemically connected by any sort of chemical bond such as covalent, ionic, coordination, chelation bonding, etc., which those skilled in the art will recognize can be synthesized on a solid-phase support. The term polymer as used herein includes those compounds conventionally called heteropolymers, i.e., arbitrarily large molecules composed of varying monomers, wherein the monomers are linked by means of a repeating chemical bond or structure. The polymers of the invention of this types are composed of subunits or monomers that can include any bi-functional organic or herteronuclear molecule including, but not limited to amino acids, amino hydroxyls, amino isocyanates, diamines, hydroxycarboxylic acids, oxycarbonylcarboxylic acids, aminoaldehydes, nitroamines, thioalkyls, and haloalkyls. In the disclosure of the present invention, the terms xe2x80x9cmonomer,xe2x80x9d xe2x80x9csubunitsxe2x80x9d and xe2x80x9cbuilding blocksxe2x80x9d will be used interchangeably to mean any type of chemical building block of molecule that may be formed upon a solid-phase support.
Various techniques for synthesizing libraries of compounds on solid-phase supports are known in the art. Solid-phase supports are typically polymeric objects with surfaces that are functionalized to bind with subunits or monomers to form the compounds of the library. Synthesis of one library typically involves a large number of solid-phase supports. Solid-phase supports known in the art include, among others, polystyrene resin beads, cotton threads, and membrane sheets of polytetrafluoroethylene (xe2x80x9cPTFExe2x80x9d).
To make a combinatorial library, solid-phase supports are reacted with a one or more subunits of the compounds and with one or more numbers of reagents in a carefully controlled, predetermined sequence of chemical reactions. In other words, the library subunits are xe2x80x9cgrownxe2x80x9d on the solid-phase supports. The larger the library, the greater the number of reactions required, complicating the task of keeping track of the chemical composition of the multiple species of compounds that make up the library. Thus, it is important to have methods and apparatuses which facilitate the efficient production of large numbers of chemical compounds, yet allow convenient tracking of the compounds over a number of reaction steps necessary to make the compounds.
One method of making combinatorial libraries is described in U.S. Pat. No. 5,510,240 to Lam et al. (xe2x80x9cLam ""240 patentxe2x80x9d), the disclosure of which is incorporated herein by reference in its entirety. More specifically, the Lam ""240 patent discloses a split and mix method of synthesizing combinatorial libraries of bio-oligomers on resin beads, in certain embodiments of which the library contains all possible combinations of monomer subunits of which the bio-oligomers are composed. Although there may be several resin beads containing the same species of bio-oligomer, each resin bead contains only one species of bio-oligomer.
Another example of a method of making combinatorial libraries on divisible solid-phase supports is described in U.S. Pat. No. 5,688,696 to Lebl (xe2x80x9cLebl ""696 patentxe2x80x9d), the disclosure of which is incorporated herein by reference in its entirety. In the method disclosed in the Lebl ""696,each of a set of predetermined species of test compounds is present on a predetermined number of solid-phase supportsxe2x80x94preferably on only onexe2x80x94and each solid-phase support has only a single species of test compound.
The use of radio-frequency identification (xe2x80x9cRFIDxe2x80x9d) chips to record the steps of library synthesis is also known. See, for example, U.S. Pat. Nos. 5,741,462, 5,770,455, and 5,751,629, as well as WO 98/15826.
A method and apparatus for synthesis of a combinatorial library using a 3-D array of reaction zones is provided in Glaxo""s WO 99/32219 (xe2x80x9cGlaxo Applicationxe2x80x9d). This application discloses stackable frames having a plurality of holes. Membranes, which act as the solid supports, are trapped between stacked frames, and these membranes are exposed at the frame holes. In an alternative embodiment, solid support beads are placed on flow-through sieves that allow flow-through of reagents around the support beads. Reagents are pumped in from the top and vacated at the bottom or, alternatively, pumped in from the bottom and vacated at the top. The apparatus disclosed allows reagents to be delivered to groups of supports in the X-Z planes or in the Y-Z planes during synthesis steps.
The Glaxo Application also employs a 3-D (X-Y-Z) array of supports. However, instead of using a containment apparatus having true wells in which solid supports are stacked, the Glaxo method employs stackable 2-D (X-Y) frames. The Glaxo Application discloses two distinct embodiments of stackable frame structures. One embodiment sandwiches a membrane between stacked frames, the frames having a plurality of holes. The membranes are solid-phase supports which are held between the frames. The frame holes expose the membranes. The membranes also have holes to allow reagents to pass through the layers of membranes and contact other membranes in the vertical xe2x80x9ccolumnxe2x80x9d of the array. Another embodiment has sieves in place of the membranes, and free solid supports are placed on each sieve between the frames. The sieves allow reagents to flow vertically from top to the bottom of the stacked 3-D array contacting a vertical column of solid-phase supports resting on sieves.
A major disadvantage with Glaxo""s apparatus and method, however, is that after the synthesis is completed, the solid supports, whether as the membrane or the solid-phase support beads suspended on the sieve, are not easily freed from the stacked array while retaining their spatial identities. The frames must be taken apart one by one to gain access to the supports and to provide some means to retain the identities of each support. This requires a burdensome additional step that makes the apparatuses disclosed less attractive for commercial production of libraries.
While methods exist in the art that can be used to produce a library of compounds, there is still a need for methods and apparatuses effective for commercial use to build a large library of compounds quickly and with a minimum of cost. Thus, there is still a need for alternative methods of synthesis that use 2-D or 3-D arrays of solid-phase support as part of the synthesis process for the purpose of commercially making large libraries of compounds efficiently.
Moreover, there is still a need for apparatuses and methods for efficiently synthesizing extremely large libraries, e.g., greater than 100,000 compounds, using 2-D or 3-D arrays as tools in the synthesis.
The present invention provides methods and apparatuses that use 2-D or 3-D array of solid-phase supports and that may be used to commercially synthesize a library of compounds. In particular a method is provided which may be commercially used to produce large libraries having between about 100,000 to 200,000 compounds. A number of embodiments of methods and apparatuses for synthesizing libraries of compounds are provided herein in accordance with the present invention.
In a first embodiment, in accordance with the present invention, a 3-D array of solid-phase supports is used to provide parallel synthesis. One embodiment of the apparatus which provides this 3-D array is a containment device which has a plurality of wells wherein discrete solid-phase supports can be placed into and stacked in a column. In another embodiment of the apparatus, a 3-D array is formed by stacking a plurality of 2-D frames which have solid-phase supports arranged in an orderly X-Y array. The frames have a plurality of holes arranged in an orderly X-Y array and solid-phase supports can be friction fitted or interlocked into these holes to temporarily hold the supports to the frame during synthesis. Alternatively, the supports can be physically attached to the frames in a manner in which, when desired, they can be easily cut from the frame. Associated with this 3-D array, specific embodiments of the apparatuses are disclosed, in accordance with the present invention, including a 3-D containment plate which has double-drilled holes, a gear-shaped solid-phase supports (xe2x80x9cgearxe2x80x9d) designed to be friction fitted or interlocked into 2-D frame holes, a lantern-shaped solid-phase supports (xe2x80x9clanternxe2x80x9d), and ring supports used in conjunction with a containment device having a plurality of wells.
A specific synthesis method is provided, which can be used with an apparatus having a 3-D arrangement of solid-phase supports, in accordance with the present invention. A preferred method provides a monomer or subunit diversity to the library compounds on the solid-phase supports between the X-Y layers in the Z direction. The method comprises: providing reagents to react with solid-phase supports in the X-Z layers, providing reagents to react with solid-phase supports in Y-Z layers, and retrieving columns of solid-phase supports, while retaining their spatial relationships.
A defining characteristic of this first method embodiment using a 3-D array of support is once the array is formed, the supports are generally not moved during the subsequent synthesis steps. Reagents for reacting with the supports are brought to the array and usually a particular reagent is delivered only to a subset of the supports in the 3-D array. Additionally, the size of the library of compound will be limited by the size of the 3-D array.
In a second method embodiment, in accordance with the present invention, the same stackable frames are used as in the first embodiment. Frames having X-Y arrays of solid-phase supports are stacked into 3-D arrays (xe2x80x9cstacksxe2x80x9d). Instead of a single 3-D array, in this second embodiment a multiple N number of stacks are formed in preparation for making a library of compounds.
In the first synthesis step, each stack numbered 1 to N is completely immersed into separate reactors 1 to N respectively, each reactor having a distinct reagent and a subunit is attached to each support in the stack. After each stack is removed from its reactor, a first randomization occurs by taking one and only one layer (frame) of each original stack, combining these layers to form a new stack. Thus, the first layer or frame from each original stack is grouped to create a first new stack, the second layer or frame from each original stack is grouped to create a second new stack. This reshuffling process is repeated until all the original frames of each old stack are transferred to a set of N number of new stacks. Then, in the second synthesis step, each new stack from 1 to N is immersed in a set of reactors, each reactor having a different reagent.
In the second randomization step, one vertical column of solid-phase supports is removed from each new stack keeping the spatial identification of the supports intact and then reassembled to make a new grouping of 3-D supports. Another vertical column of supports is removed from each new stack and regrouped to another grouping of 3-D supports. This process is repeated until all the supports in the new stack arrays have been regrouped into a N number of new 3-D arrays. In this regrouping, randomization step, only one vertical column of supports is taken from each new stack to make a new grouping of supports. In the third and final synthesis step, the new groupings of supports are each put into separate 1 to N reactors, each reactor having a different reagent.
The apparatuses used with this second embodiment are the same as used in the first embodiment. A preferred embodiment of the frame and solid-phase support is a 2-D frame having a plurality of holes arranged in an X-Y rectangular order. A preferred apparatus comprises gears or lanterns friction fitted or interlocked into the plurality of holes. Additionally, reactors having a capacity large enough for immersion of 3-D stacks are needed.
The defining characteristics of this second embodiment are: (a) many N number of 3-D original stacks are formed; (b) the original stacks do not have solid supports which have a subunit attached in contrast to embodiment one; (c) the solid supports are disturbed from the original 3-D arrays because the supports are moved during the synthesis process when the frames are reshuffled and vertical columns of supports are regrouped; and (d) every solid support in each 3-D stack is completely immersed in the reagent during a synthesis step because the stack is brought to the reagents/reactors. The second embodiment lends itself to large scale production of libraries of compounds because the final number of unique compounds is based on the number N of original stacks made.
The third embodiment, in accordance with the present invention, uses 2-D frames in a xe2x80x9csort and combinexe2x80x9d method of synthesis. There is no stacking of the frames into a 3-D array. Instead, the 2-D frames are split during synthesis of the combinatorial library. The method of this third embodiment can be implemented by automation since no rods are required and may be used to generate large libraries, having between about 100,000 to 200,000 compounds.
In this method, a Q number of 2-D frames is chosen. The 2-D frames have rows and columns. Solid supports are placed into reagents for a first synthesis step. Solid supports thus reacted with a single subunit are placed into the frame holes such that the frame has columns of supports which have the same subunit, but between columns, there is a diversity of subunits. This placement provides the first randomization. Each Q number of frames is initially identically prepared. Next, in a second synthesis step, the Q frames are placed into 1 to Q reactors, each having a different reagent. After removal from the reactors, the Q number of frames are split up into subframes to provide the second randomization. M new groups of subframes are regrouped by taking one and only one subframe from each original frame. M represents the number of subframes a frame has been split into. The M new groups of subframes, each are immersed into 1 to M reactors, each reactor having a different reagent. After final synthesis the supports are detached from the subframes and placed into a labeled cleavage plate.
The total number of unique compounds in the library is Qxc3x97Mxc3x97N, where N is the number of columns present in the original 2-D frames, and Q is arbitrarily chosen. The size of the library will be controlled by choice of three variables Q, M and N.
The preferred apparatuses used in this embodiment are 2-D frames. Solid-phase supports such as gears are friction fitted or interlocked into the plurality of holes in the frame. The additional feature of the frame is that it must be easily splittable into subframes. Reactors are need which have capacity for accepting groups of subframes. Additionally, in accordance with a preferred embodiment of the present invention, a 2 row subframe having a RIFD chip is disclosed.
The defining characteristics of this third method embodiment are: (a) user choice of the number of frames Q to use in the synthesis; (b) the solid supports are disturbed from the original 2-D arrays because the supports are moved during the synthesis process when the frames are split and regrouped; and (c) every solid support in each 2-D frame or 2-D subframe is completely immersed in the reagent during a synthesis step because the frame or group of frames is brought to the reagents/reactors. The third embodiment lends itself to large scale production of libraries of compounds because the final number of unique compounds is based on the number Q of original frames used.
All three method embodiments use 2-D or 3-D arrays of supports held in frames to facilitate parallel synthesis on solid-phase supports and to provide spatial identification and thus the synthesis history of the compound produced on a particular support.
There is interchangeability of apparatuses used in the various embodiments described above, in accordance with the present invention. For example, the supports, frames, rods and devices for removing the supports from the frames are interchangeable. A gear design of solid support for use with 2-D frames is provided in accordance with the present invention. A new embodiment of a 3-D containment plate having double-drilled holes and RFID chip is provided in accordance with the present invention.